Biomedical subjects

Ellen L Zechner

Publications and source records attributed to Ellen L Zechner.

11 recordsLinked to original sources

PubMed2006

General mutagenesis of F plasmid TraI reveals its role in conjugative regulation.

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies approximately 100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.

Conjugation, Genetic↗

PubMed2006

In vitro biofilm formation of commensal and pathogenic Escherichia coli strains: impact of environmental and genetic factors.

Our understanding of Escherichia coli biofilm formation in vitro is based on studies of laboratory K-12 strains grown in standard media. However, pathogenic E. coli isolates differ substantially in their genetic repertoire from E. coli K-12 and are subject to heterogeneous environmental conditions. In this study, in vitro biofilm formation of 331 nondomesticated E. coli strains isolated from healthy (n = 105) and diarrhea-afflicted children (n = 68), bacteremia patients (n = 90), and male patients with urinary tract infections (n = 68) was monitored using a variety of growth conditions and compared to in vitro biofilm formation of prototypic pathogenic and laboratory strains. Our results revealed remarkable variation among the capacities of diverse E. coli isolates to form biofilms in vitro. Notably, we could not identify an association of increased biofilm formation in vitro with a specific strain collection that represented pathogenic E. coli strains. Instead, analysis of biofilm data revealed a significant dependence on growth medium composition (P < 0.05). Poor correlation between biofilm formation in the various media suggests that diverse E. coli isolates respond very differently to changing environmental conditions. The data demonstrate that prevalence and expression of three factors known to strongly promote biofilm formation in E. coli K-12 (F-like conjugative pili, aggregative adherence fimbriae, and curli) cannot adequately account for the increased biofilm formation of nondomesticated E. coli isolates in vitro. This study highlights the complexity of genetic and environmental effectors of the biofilm phenotype within the species E. coli.

Bacteremia↗

PubMed2006

Synergistic effects in mixed Escherichia coli biofilms: conjugative plasmid transfer drives biofilm expansion.

Bacterial biofilms, often composed of multiple species and genetically distinct strains, develop under complex influences of cell-cell interactions. Although detailed knowledge about the mechanisms underlying formation of single-species laboratory biofilms has emerged, little is known about the pathways governing development of more complex heterogeneous communities. In this study, we established a laboratory model where biofilm-stimulating effects due to interactions between genetically diverse strains of Escherichia coli were monitored. Synergistic induction of biofilm formation resulting from the cocultivation of 403 undomesticated E. coli strains with a characterized E. coli K-12 strain was detected at a significant frequency. The survey suggests that different mechanisms underlie the observed stimulation, yet synergistic development of biofilm within the subset of E. coli isolates (n = 56) exhibiting the strongest effects was most often linked to conjugative transmission of natural plasmids carried by the E. coli isolates (70%). Thus, the capacity of an isolate to promote the biofilm through cocultivation was (i) transferable to the K-12 strain, (ii) was linked with the acquisition of conjugation genes present initially in the isolate, and (iii) was inhibited through the presence in the cocultured K-12 strain of a related conjugative plasmid, presumably due to surface exclusion functions. Synergistic effects of cocultivation of pairs of natural isolates were also observed, demonstrating that biofilm promotion in this system is not dependent on the laboratory strain and that the described model system could provide relevant insights on mechanisms of biofilm development in natural E. coli populations.

Bacteremia↗

PubMed2005

Unsaturated fatty acids are inhibitors of bacterial conjugation.

This report describes a high-throughput assay to identify substances that reduce the frequency of conjugation in Gram-negative bacteria. Bacterial conjugation is largely responsible for the spread of multiple antibiotic resistances in human pathogens. Conjugation inhibitors may provide a means to control the spread of antibiotic resistance. An automated conjugation assay was developed that used plasmid R388 and a laboratory strain of Escherichia coli as a model system, and bioluminescence as a reporter for conjugation activity. Frequencies of conjugation could be measured continuously in real time by the amount of light produced, and thus the effects of inhibitory compounds could be determined quantitatively. A control assay, run in parallel, allowed elimination of compounds affecting cell growth, plasmid stability or gene expression. The automated conjugation assay was used to screen a database of more than 12,000 microbial extracts known to contain a wide variety of bioactive compounds (the NatChem library). The initial hit rate was 1.4 %. From these, 48 extracts containing active compounds and representing a variety of organisms and extraction conditions were subjected to fractionation (24 fractions per extract). The 52 most active fractions were subjected to a secondary analysis to determine the range of plasmid inhibition. Plasmids R388, R1 and RP4 were used as representatives of a variety of plasmid transfer systems. Only one fraction (of complex composition) affected transfer of all three plasmids, while four other fractions were active against two of them. Two separate compounds were identified from these fractions: linoleic acid and dehydrocrepenynic acid. Downstream analysis showed that the chemical class of unsaturated fatty acids act as true inhibitors of conjugation.

Conjugation, Genetic↗

PubMed2004

Concomitant reconstitution of TraI-catalyzed DNA transesterase and DNA helicase activity in vitro.

TraI protein of plasmid R1 possesses two activities, a DNA transesterase and a highly processive 5'-3' DNA helicase, which are essential for bacterial conjugation. Regulation of the functional domains of the enzyme is poorly understood. TraI cleaves supercoiled oriT DNA with site and strand specificity in vitro but fails to initiate unwinding from this site (nic). The helicase requires an extended region of adjacent single-stranded DNA to enter the duplex, yet interaction of purified TraI with oriT DNA alone or as an integral part of the IncF relaxosome does not melt sufficient duplex to load the helicase. This study aims to gain insights into the controlled initiation of both TraI-catalyzed activities. Linear double-stranded DNA substrates with a central region of sequence heterogeneity were used to trap defined lengths of R1 oriT sequence in unwound conformation. Concomitant reconstitution of TraI DNA transesterase and helicase activities was observed. Efficient helicase activity was measured on substrates containing 60 bases of open duplex but not on substrates containing < or =30 bases in open conformation. The additional presence of auxiliary DNA-binding proteins TraY and Escherichia coli integration host factor did not stimulate TraI activities on these substrates. This model system offers a novel approach to investigate factors controlling helicase loading and the directionality of DNA unwinding from nic.

Base Sequence↗

PubMed2004

The Horizontal Gene Pool: an ESF workshop summary.

The European Science Foundation (ESF) funds a limited number of exploratory workshops each year that enable scientists to meet and develop plans for a program of integrated research which would benefit from a coordinated European effort. In summer 2003, the Standing Committee for Life and Environmental Sciences (LESC) sponsored such a workshop called The Horizontal Gene Pool: The Functional Role of Mobile Genetic Information--How Bacteria Perceive, Sample, and Utilize Genetic Elements in evolution and Local Adaptation. The workshop took place at St. Catherine's College, Oxford, UK. Its purpose was to identify how recent advances in the application of genomics and microbial ecology can be harnessed to determine the genetic mechanisms that underpin the biological role of the horizontal gene pool. Scientific excellence at the workshop was contributed by senior scientists and young investigators from research institutes located in nine European countries.

Bacteria↗

PubMed2003

Extent of single-stranded DNA required for efficient TraI helicase activity in vitro.

The IncF plasmid protein TraI functions during bacterial conjugation as a site- and strand-specific DNA transesterase and a highly processive 5' to 3' DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5' to 3' unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5' tail of single-stranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of single-stranded DNA of defined length was created and its structure verified. We found that substrates containing >or=27 nucleotides of single-stranded DNA 5' to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands.

Base Sequence↗

PubMed2003

Development and maturation of Escherichia coli K-12 biofilms.

The development and maturation of E. coli biofilms in flow-chambers was investigated. We found that the presence of transfer constitutive IncF plasmids induced biofilm development forming structures resembling those reported for Pseudomonas aeruginosa. The development occurred in a step-wise process: (i). attachment of cells to the substratum, (ii). clonal growth and microcolony formation, and (iii). differentiation into expanding structures rising 70-100 microm into the water phase. The first two steps were the same in the plasmid-carrying and plasmid-free strains, whereas the third step only occurred in conjugation pilus proficient plasmid-carrying strains. The final shapes of the expanding structures in the mature biofilm seem to be determined by the pilus configuration, as various mutants affected in the processing and activity of the transfer pili displayed differently structured biofilms. We further provide evidence that flagella, type 1 fimbriae, curli and Ag43 are all dispensable for the observed biofilm maturation. In addition, our results indicate that cell-to-cell signalling mediated by autoinducer 2 (AI-2) is not required for differentiation of E. coli within a biofilm community. We suggest on the basis of these results that E. coli K-12 biofilm development and maturation is dependent on cell-cell adhesion factors, which may act as inducers of self-assembly processes that result in differently structured biofilms depending on the adhesive properties on the cell surface.

Bacterial Adhesion↗

PubMed2003

Species-specific identification of campylobacters by partial 16S rRNA gene sequencing.

Species-specific identification of campylobacters is problematic, primarily due to the absence of suitable biochemical assays and the existence of atypical strains. 16S rRNA gene (16S rDNA)-based identification of bacteria offers a possible alternative when phenotypic tests fail. Therefore, we evaluated the reliability of 16S rDNA sequencing for the species-specific identification of campylobacters. Sequence analyses were performed by using almost 94% of the complete 16S rRNA genes of 135 phenotypically characterized Campylobacter strains, including all known taxa of this genus. It was shown that 16S rDNA analysis enables specific identification of most Campylobacter species. The exception was a lack of discrimination among the taxa Campylobacter jejuni and C. coli and atypical C. lari strains, which shared identical or nearly identical 16S rDNA sequences. Subsequently, it was investigated whether partial 16S rDNA sequences are sufficient to determine species identity. Sequence alignments led to the identification of four 16S rDNA regions with high degrees of interspecies variation but with highly conserved sequence patterns within the respective species. A simple protocol based on the analysis of these sequence patterns was developed, which enabled the unambiguous identification of the majority of Campylobacter species. We recommend 16S rDNA sequence analysis as an effective, rapid procedure for the specific identification of campylobacters.

Animals↗

PubMed2002

TraG-like proteins of DNA transfer systems and of the Helicobacter pylori type IV secretion system: inner membrane gate for exported substrates?

TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.

Acid Anhydride Hydrolases↗

PubMed2002

Transmission of Campylobacter hyointestinalis from a pig to a human.

We report on a case of human gastroenteritis caused by the pathogen Campylobacter hyointestinalis. Recurrent watery diarrhea and intermittent vomiting were the most significant symptoms of the previously healthy patient. Whole-cell protein electrophoresis and 16S rRNA gene sequencing were used to identify this Campylobacter species. Investigation of the patient's surroundings led to the recovery of a second C. hyointestinalis strain originating from porcine feces. Subsequent typing of the human and the porcine isolates by pulsed-field gel electrophoresis revealed similar macrorestriction profiles, indicating transmission of this pathogen.

Aged↗